Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the oldest known human maladies. Yet this disease is still one of the major causes of mortality, as almost 2 million people die each year from this disease. Despite the widespread use of an attenuated live vaccine and several antibiotics, there is more TB than ever before, requiring new vaccines, drugs and more specific and rapid diagnostics. Mtb is a facultative intracellular pathogen that replicates in macrophages and extracellularly in lung cavities. During infection, Mtb is exposed to diferent environments and stres conditions to which it must adapt in order to survive and multiply. Iron deficiency is one of those conditions. As is the case for most living organisms, Mtb requires iron as cofactor for enzymes that are involved in essential functions, including respiration, DNA replication and defense against toxic oxidative stress. Thus, if ways can be found to interfere with Mtb's ability to acquire iron, this information could be useful in designing new anti-tubercular therapies. The specific aims of the proposal are first, to understand how the major pathway of Mtb iron acquisition, using siderophores, functions. This includes learning how iron complexed to siderophores is imported into Mtb and how iron is then released in a form that can be used by the cell for its essential functions. Because iron acquisition is essential, pathogens do not usually rely in a single method to get this nutrient. We found that Mtb can use heme and hemoglobin as iron source independently of siderophores. We will explore the mechanisms for iron acquisition from heme and the possible role of heme utilization in Mtb's virulence. The 2nd aim seeks to understand why IdeR, the major regulator of iron uptake and storage is essential, using a combination of in vitro and in vivo approaches. The 3rd aim wil use the information obtained in the first two aims and earlier work to develop new live attenuated, safe vaccine strains that could be used to prevent TB in the general population, as well as immunocompromised individuals.